Formation and characterization of nitric oxide-dependent anammox granules

As a sustainable nitrogen removal technology, anammox granular sludge (AnGS) has been widely used in treating various wastewater due to its high nitrogen removal rate and resilience to hydraulic and loading disturbances. However, the relatively slow granulation rate of AnGS is a major bottleneck for its broader applications. Nitric oxide (NO), a key nitrogen-cycle intermediate and microbial signaling molecule, can directly serve as an electron acceptor for anammox bacteria, potentially enhancing metabolic activity. Furthermore, direct gas sparging provides shear force to AnGS that can facilitate biomass aggregation and granule formation. This study demonstrated that NO sparging could accelerate AnGS granulation by coupling metabolic stimulation with shear-induced aggregation. Through long-term cultivation and adaption, the NO-fed reactor (R1) achieved highly efficient and stable NO removal, with a maximum NO removal efficiency of 93.6%. In addition, NO sparging increased the proportions of large-size (∼38%) and medium-size (∼48%) granules in R1, compared to 30% and 25%, respectively, in the control group (R2) without NO sparging (2000 ppm). The AnGS in R1 exhibited enhanced structural stability and elevated metabolic activity. 'Candidatus Kuenenia' was identified as the most abundant in medium-size granules (with a relative abudance of 5.5%). Metatranscriptomic analysis further validated that NO sparging upregulated genes involved in nitrogen metabolisms (e.g., hdh, hzsB) and fundamental energy metabolism (e.g., cooS) within the granular sludge system. Overall, this study demonstrates that NO can be leveraged as a dual metabolic and physical control strategy to accelerate anammox granulation, providing new insights for the integrated management of gaseous and aqueous pollutants.